The correct formation of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides, including many proteins of pharmacological importance. Great progress has been made in the last few years in understanding the mechanism of disulfide oxidation in vivo, but the mechanism of reduction and isomerization of incorrectly formed disulfides is less clear. We have recently succeeded in the reconstitution of the isomerization/reduction pathway in vitro. This reconstitution opens up the way to extensive biochemical and mechanistic analysis of the process of disulfide correction. DsbC is a periplasmic protein that is thought to isomerize misfolded proteins. In order to be active, DsbC must be kept reduced in the very oxidizing periplasmic environment. DsbD reduces DsbC. DsbD in turn is reduced by the cytoplasmic thioredoxin. We aim to solve the interesting and long-standing topological puzzle of how disulfide bonds are transported through membranes. We have succeeded in defining the direction of electron flow between the individual domains of DsbD, and between DsbD, DsbC, and thioredoxin. Now we must determine how disulfides actually are transported through the membrane. This transport process can be thought of as an outward flow of electrons or an inward flow of disulfides. We will test two models for DsbD action, one where DsbD works via simple disulfide exchange reactions, and the second where cofactor dependent electron transport is also involved. To do this we focus on examining electron flow through the beta domain of DsbD which is the domain that spans the membrane. This domain appears to contain an iron cofactor. We will examine the role of this iron in DsbD catalyzed electron transport. Efforts to define in vivo substrates for DsbC and DsbG will give us tools to eventually address the long-standing question of how disulfide isomerization actually occurs in vivo. Overall, we aim to understand in detail the function of the DsbC-DsbD disulfide isomerase/reductase machine and in particular how disulfides are transported across membranes by the DsbD protein.